It is proposed to characterize and correlate the structural, motional and enzymatic alterations in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which accompany its binding to the cytoplasmic N-terminal region of band 3 protein in the human RBC membrane. Electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) techniques will be utilized in conjunction with recently synthesized high resolution [15N, 2H] spin labeled analogues of coenzyme NAD+ and active site reactive maleimide spin label to define changes in the local and tetrameric structure of GAPDH and changes in rotational frequencies upon binding to intact ghost membranes and purified cleavage fragments of band 3. These results will be correlated with Lineweaver-Burke plots of kinetic data obtained from soluble and membrane bound GAPDH. Band 3 will be labeled at the extracellular surface in intact RBC and ghost membrane preparations with newly designed and synthesized spin label reagents. Rotational diffusion coefficients DII and DI for motions about the membrane normal axis and an axis perpendicular respectively will be determined by direct computer simulation of experimental ST-EPR spectra. These investigations may be of importance in subsequent characterizations of abnormal RBC membranes encountered in diseases such as Duchenne and myotonic dystrophies and diabetes. Spin labeled ATP analogues will be employed to examine the functional relationship between membrane bound glycolytic enzymes GAPDH, phosphoglycerate kinase (PGK) and the Na+, K+-ATPase. Experiments are proposed to determine the characteristics of membrane associated compartments of ATP which have been hypothesized. The proposed research will provide detailed insight into the production and mode of utilization of ATP in the RBC as well as establish methodologies for subsequent studies of membrane protein-protein interactions.